Enhanced RNA replication and pathogenesis in recent SARS-CoV-2 variants harboring the L260F mutation in NSP6

Abstract

The COVID-19 pandemic has been driven by SARS-CoV-2 variants with enhanced transmission and immune escape. Apart from extensive evolution in the Spike protein, non-Spike mutations are accumulating across the entire viral genome and their functional impact is not well understood. To address the contribution of these mutations, we reconstructed genomes of recent Omicron variants with disabled Spike expression (replicons) to systematically compare their RNA replication capabilities independently from Spike. We also used a single reference replicon and complemented it with various Omicron variant Spike proteins to quantify viral entry capabilities in single-round infection assays. Viral entry and RNA replication were negatively correlated, suggesting that as variants evolve reduced entry functions under growing immune pressure on Spike, RNA replication increases as a compensatory mechanism. We identified multiple mutations across the viral genome that enhanced viral RNA replication. NSP6 emerged as a hotspot with a distinct L260F mutation independently arising in the BQ.1.1 and XBB.1.16 variants. Using mutant and revertant NSP6 viral clones, the L260F mutation was validated to enhance viral replication in cells and increase pathogenesis in mice. Notably, this mutation reduced host lipid droplet content by NSP6. Collectively, a systematic analysis of RNA replication of recent Omicron variants defined NSP6's key role in viral RNA replication that provides insight into evolutionary trajectories of recent variants with possible therapeutic implications.

Fig 1. Recent Omicron variants have higher replication than early ones.

A and B) Consensus non-Spike (A) and Spike (B) mutations (>95% of sequences at time of emergence) in all dominant SARS-CoV-2 variants post-Delta. BA.2 fixed mutations are indicated in red. C) RNA replication measurement of each variant paired with its concordant Spike expression vector using SARS-CoV-2 Spike replicons. The data is plotted as mean +/- SD of two independent biological replicates conducted in triplicate. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 by two-sided Student’s T-test. NTD: N-terminal domain; RBD: receptor binding domain; FCS: furin cleavage site; VAT: Vero cells stably expressing ACE2 and TMPRSS2; A549-ACE2^h: A549 cells stably expressing high levels of ACE2.

Fig 2. RNA replication and entry negatively correlate for Omicron variants.

A) Schematic of replicon experiments to measure RNA replication and entry. B and C) Normalized RNA replication and normalized entry for all variants were measured in VAT (B) and A549-ACE2^h (C) cells and graphed on a scatter plot. Data is plotted as mean +/- SD of two independent biological replicates each conducted in triplicate. VAT: Vero cells stably expressing ACE2 and TMPRSS2; A549-ACE2^h: A549 cells stably expressing high levels of ACE2.

Fig 3. NSP6 L260F mutation enhances RNA replication and reduces LD content in infected cells.

A) NSP6 mutations in Omicron variants. BA.2 fixed mutations are indicated in red. B) RNA replication measurement of indicated variants in VAT cells. Data is plotted as mean +/- SD of three independent biological replicates each conducted in duplicate. C) Quantification of the relative LD mean fluorescent intensity (MFI) per dsRNA positive cells in images shown in S2 Fig using box and whiskers plot, and comparisons were made as indicated by two-sided Student’s T-test. D) Immunofluorescence staining of HuH-7.5 cells stably expressing indicated NSP6 proteins and stained for LD, PLIN2, and NSP6. E, F, and G) Quantification of the relative LD MFI, LD number, and average LD size per NSP6-positive cells in images shown in D using box and whiskers plot, and comparisons were made as indicated by two-sided Student’s T-test. For panels using box and whiskers plots, interquartile range (IQR) of boxplot is between 25th and 75th percentiles and center line indicates median value. Whiskers of boxplot is extended to the maxima and minima. Maxima is the largest value and minima is the smallest value in the dataset. LD, lipid droplet. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 by two-sided Student’s T-test.

Fig 4. NSP6 L260F is critical for viral infection in vitro and pathogenesis in vivo.

A) Plaque morphology of indicated viruses in VAT cells at 48 hours post-infection. The images were pseudocolored to black and white for optimal visualization. B) Particle production and intracellular viral RNA abundance were measured by plaque assay and RT-qPCR, respectively, at 24 and 48 hours post-infection in A549-ACE2^h cells for indicated viruses. Data is plotted as mean +/- SD for three independent biological replicates each conducted in duplicate. C) Particle production was measured by plaque assay at 24, 48, and 72 hours post-infection in VAT cells for indicated viruses. Data is plotted as mean +/- SD of one representative biological replicate conducted in triplicate. D) Schematic representation of the in vivo experiment to measure the replication and pathogenesis of BA.5 and BA.5 NSP6 F260L viruses (10⁵ PFU) in K18-hACE2 mice. n=10 per virus group per time-point. Clip art was created with BioRender.com with permission. E) Animal weight is plotted over the study time course as mean +/- SEM. BA.5 group animals all succumbed to infection at 7 days post-infection and therefore weight data are only available until that time-point. Survival was determined based on the humane endpoint (HE) of 20% weight loss and is plotted as a Kaplan-Meier curve. F and G) Particle production and intracellular viral RNA abundance were measured by plaque assay and RT-qPCR, respectively, at 5 (F) and 14 (G) days post-infection. For mice not reaching the end of the study, their tissues were collected when they reached the HE. Data is plotted as mean +/- SD. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 by two-sided Student’s T-test.